1. Field of the Invention
The invention is a method for producing and/or using arrays which contain a multiplicity of compounds for the purpose of screening to identify molecules with desirable properties. The various compounds forming the elements of the array can be synthesized in place using combinatorial synthesis schemes, or pre-synthesized compounds can be incorporated into the array. Applications to genetic screening, in vitro diagnostics, and drug discovery are anticipated.
2. Discussion of the Prior Art
Current trends in medical diagnostic testing and pharmaceutical research are toward conducting a large number of tests concurrently on a single device. For example, one such device has been called a DNA "chip" for sequence analysis. A DNA chip contains a large number (thousands) of unique DNA molecules (probes) immobilized on a flat surface in the form of an array (e.g. checker board). The company Affymax uses a photo-lithographic method to produce DNA chips (Fodor, S.P.A., et. al., Science, 1991, 251, 767-773). Another approach is to use pre-synthesized molecules which are applied and immobilized to a suitable substrate (e.g. microporous membrane).
For example, an unknown sample of DNA (target) is applied to the chip and a hybridization pattern is formed. The pattern is indicative of the strength of interaction between the target DNA and the various immobilized probes and can yield sequence information. When the sequence of the target DNA is not known the technology is generally referred to as sequencing by hybridization (SBH) as described in U.S. Pat. No. 5,202,231. In other applications where the sequence of the target is known and detection is directed at identification of a change associated with a disease state the method is commonly referred to as "re-sequencing" or allele specific oligonucleotide hybridization.
Another trend in the arena of pharmaceutical drug discovery is known as combinatorial synthesis. In this case a large number of similar compounds are synthesized and simultaneously screened for the desired biological response, for example binding to a receptor molecule. When one or more candidate compounds in the combinatorial bank are discovered they are synthesized in larger quantities for further testing. Molecules of interest include peptides, nucleic acids as well as drug compounds synthesized by standard organic chemical methods or other novel methods for drug discovery.
Finally, in the area of in vitro diagnostics there is a need for panel assays where several tests are run concurrently on a given sample using an array of immobilized binding agents. An example of such array immunoassay devices is described in U.S. Pat. Nos. 4,591,570 and 4,829,010. Pre-manufactured compounds, such as mono-clonal antibodies, are used to make arrays whose elements have particular binding properties for diagnostic analysis. In principle a patient test sample can be simultaneously analyzed for the presence or absence of several molecules, i.e. analytes. Further, the levels of the various analytes can be measured simultaneously by quantitative analysis of the signals developed at each site of the array. In other applications there is a need for graphic symbols that can be visually analyzed to determine the presence or absence of a single analyte in a patient test sample. For the present invention, the graphic symbol can be thought of as an array of individual elements that are spatially arranged to yield a graphic symbol as a result of the detection process. In this case, the size of the array elements determine the "grain" of the graphic symbol.
Thus, there is a need for methods to produce and concurrently test multiple compounds or binding agents in the form of an array. An additional requirement is the need for high density devices (i.e. high spatial density) so that the large numbers of compounds are presented in a package of reasonable size. For example, a device that contains all possible 8-mer DNA sequences composed of the 4 DNA bases, A (adenine), T (thymine), G (guanine) and C (cytosine) requires 48=65536 different compounds. If each element (i.e. a zone of immobilized binding agent) was a square only 1 millimeter (1 mm=0.1 cm) in size, an array of 65536 elements would be 10 inches on a side. Clearly, such devices would be difficult to manipulate and would require relatively large amounts of the test sample to be spread evenly over the array surface. A 0.1 mm thick layer of test sample spread on a 10.times.10 inch area amounts to about 6.5 ml (ml=milliliter=6.5.times.10-3 liter). Since most test samples are of biological origin, they are typically very expensive, difficult to prepare and in short supply. Examples of test samples are PCR products or purified drug receptors which are typically available in microliter quantities-1000 times less than in the above example. In most cases, DNA synthesis requires the use of expensive components, phosphoramidite DNA synthesis being a case in point, so surface area of the array is also important during the manufacturing step.
Thus, the smaller the size of the array elements involved in the synthesis the more economical the device will be to produce and use. Several methods exist to create chips with large numbers of different sequences but often result in devices with large features, large physical size and, hence, low spatial density. For example, one method uses disks or channels to produce arrays of probe DNA's using standard DNA synthesis chemistry (see for example, Williams, et. al., Nucleic Acids Research 1994, 22, 1365 or Southern et. al., Nucleic Acids Research, 1994, 22, 1368 and references therein). The drawback of this method is that small feature size is not obtained.
Another method of making DNA chips is to use pre-synthesized probe DNA's and a printing device to allow application of the various compounds. The probes are applied to the chip with a pin or a pipette in the pattern of an array and immobilized by any of a variety of techniques such as adsorption or covalent linkage. An example of such DNA arrays is described in Stimpson et. al. Proc. Natl. Acad. Sci. USA Vol. 92, pp. 6379-6383, July 1996. Since elements of the array are formed by the application of a DNA solution to the surface of the array the process is relatively slow.
One method to produce high density chips uses photo-lithography (Pease et. al., Proc. Natl. Acad. Sci. USA Vol. 91, pp. 5022, 1994). One drawback to this method is that it relies on a new DNA synthesis chemistry as opposed to the standard phosphoramidite chemistry used in commercial DNA synthesizers. The technology feeds off the methods evolved in the electronics industry and therefore has some of the same requirements, vis, accurate positioning to micron scales, clean room requirements and the use of multiple photo-masks to define the array pattern. Although electronic "chips" (for example an Intel Pentium.RTM. microprocessor) are mass produced economically, they are typically too expensive to be used as a disposable element, as is needed with a DNA chip.
Another drawback of such chips is the use of a solid impermeable support material like a glass slide or cover slip. As a result, only the very small amount of material immobilized on the surface of the solid chip is used to capture target molecules. An improvement is described using porous silicon or channel glass whereby hybridization reactions occur within the three-dimensional volumes of porous silicon dioxide of channel array glass rather than two-dimensional surface areas (Beattie, K. L., The 1994 San Diego Conference: The Genetic Revolution).
Unfortunately, all the array fabrication methods mentioned above suffer from a common limitation, i.e., each element of each array is a unique synthesis or an application step. This is true even when array elements or entire arrays are simply duplicated or produced "in parallel", or more accurately, concurrently. Since each element is a unique synthesis or application there is a chance for variation between corresponding elements on different arrays or, for that matter, duplicated elements on the same array. When multiple arrays are produced concurrently it is carried out in a two dimensional fashion, i.e., arrays are produced next to one another in a two dimensional X-Y plane, where X and Y refer to the two degrees of spatial freedom. In a photo-lithographic process, increasing the number of chips on a wafer (the substrate on which multiple arrays are produced) results in an increase in surface area which increases demand on the chemicals used in photo-chemistry (assuming no change in chip size).